Jung_2011_Mol.Pharmacol_80_60

The endocannabinoid 2-arachidonoyl-sn-glycerol (2-AG) is produced through hydrolysis of 1,2-diacyl-sn-glycerol (DAG), which is catalyzed by DAG lipase (DGL). Two DGL isoforms have been molecularly cloned, but their respective roles in endocannabinoid signaling have not been fully elucidated. Here, we report that DGL-alpha and DGL-beta may contribute to all-trans-retinoic acid (RA)-induced neurite outgrowth in neuroblastoma Neuro-2a cells through distinct mechanisms. RA-induced differentiation of Neuro-2a cells was associated with elevations of cellular 2-AG levels and DGL activity, which were accompanied by temporally separated transcription of DGL-alpha and DGL-beta mRNA. Knockdown of either DGL-alpha or DGL-beta expression attenuated neurite outgrowth, which indicates that both isoforms contribute to neuritogenesis. Immunostaining experiments showed that DGL-beta is localized to perinuclear lipid droplets, whereas DGL-alpha is found on plasma membranes. After RA-induced differentiation, both DGL-alpha- and DGL-beta-green fluorescent protein were distributed also in neurites but in distinguishable patterns. Overexpression of either DGL-alpha or DGL-beta increased the number of neurite-bearing cells, but DGL-beta caused substantially larger morphological changes than DGL-alpha did. Finally, the cannabinoid-1 antagonist rimonabant (1 muM) inhibited DGL-alpha-induced neuritogenesis, whereas it had no such effect on DGL-beta-induced morphological differentiation. The results indicate that RA-induced DGL expression is required for neurite outgrowth of Neuro-2a cells. The findings further suggest that DGL-alpha and -beta may regulate neurite outgrowth by engaging temporally and spatially distinct molecular pathways.